Traumatic brain injury (TBI) and Alzheimer's disease (AD) result in long-term behavioral deficits and brain neurodegeneration. There are no effective therapeutic agents for TBI or AD; therefore, advances in mechanism- based understanding of these brain disorders can identify new drug targeting approaches. Significantly, cathepsin B has been shown as a novel mechanism participating in behavioral dysfunctions and neuropathology of TBI and AD. The premise for the cathepsin B mechanism is that (a) cathepsin B is elevated in TBI and AD patients, (b) knockout (KO) of the cathepsin B gene in TBI and AD mouse models improves deficits in behavioral dysfunctions, respectively, and (c) cathepsin B KO reduces brain neuropathology. TBI and AD results in lysosomal leakage and redistribution of cathepsin B from lysosomes to the cytosol to result in cell death and activation of inflammatory IL-1? in brain. These findings lead to the hypothesis that cytosolic cathepsin B participates in the pathogenesis of TBI and AD. To test this hypothesis, it will be ideal to inhibit the pathogenic cytosolic cathepsin, without affecting its normal lysosomal function, with pH selective inhibitors as molecular probes. Our data shows that cathepsin B displays different peptide cleavage properties at neutral cytosolic pH compared to lysosomal acidic pH. These differential cleavage properties support the development of selective substrates and peptide inhibitors of cytosolic compared to lysosomal cathepsin B. The goal of this project will be to develop pH selective inhibitors of neutral cytosolic cathepsin B, compared to acidic lysosomal cathepsin B, as molecular probes for evaluation of the hypothesized pathogenic role of cytosolic cathepsin B during cellular lysosomal leakage which leads to neurodegeneration and behavioral deficits of TBI and AD. Aim 1 will assess the selective cleavage properties of cathepsin B at neutral and acidic pHs, achieved by global 'Multiplex Substrate Profiling Mass Spectrometry (MSP-MS) and positional scanning using a synthetic combinatorial library (PSSCL), for design and testing of pH selective peptide substrates. Aim 2 will utilize pH selective assays of cathepsin B to identify natural product inhibitors, achieved by screening collections of marine and terrestrial natural products, and assessing selectivity and potency. Aim 3 will develop peptidic inhibitors of cathepsin B, achieved by modifying pH selective peptide substrates with AOMK, CMK, or VS groups; selectivity and potency will be assessed. Peptidic and natural product inhibitors will be assessed for effects on cell death and IL-1? levels during A?- and H2O2-induced lysosomal leakage in neurons and glial cells. Aim 4 will characterize in vivo lysosomal leakage of cathepsin B in TBI and AD mouse models with respect to time-course, brain regions, and neuronal and glial cells. Cathepsin B inhibitors, known and newly developed inhibitors, will be given before and during lysosomal leakage for evaluation of improvements in behavioral deficits and neuropathology. The novel inhibitors will advantageously target pathogenic cytosolic cathepsin B, rather than normal lysosomal cathepsin B, for future development of TBI and AD therapeutics.